Detection of human IFN-gamma and TNF-a cytokine-producing human CD4 T cells using the Attune® Acoustic Focusing Cytometer.
Red blood cell lysed peripheral blood mononuclear cells were left unstimulated (left column) or stimulated (right column) for 5 hours with phorbol myristate acetate (PMA) and ionomycin in the presence of brefeldin A. Cells were then fixed and permeabilized using the FIX & PERM® Cell Permeabilization Kit (Cat. no. GAS003 or GAS004) and stained intracellularly with anti-human CD4 APC (Cat. no. MHCD0405) and either anti-human interferon gamma (IFN-gamma) FITC (Cat. no. MHCIFG01) (A) or anti-human tumor necrosis factor alpha (TNF-a) FITC (Cat. no. A18469) (B). Samples were collected using the Attune® Acoustic Focusing Cytometer (blue/red) with 488 nm excitation and 530/30 nm bandpass emission filter to detect FITC. 633 nm excitation and 660/20 nm bandpass emission filter were used to detect APC.